In early toxicity tests of chemicals, the measured endpoint was acute toxicity. Over the past ten years, toxicity testing has expanded to include measures of sub-chronic toxicity including cancer, immune suppression, developmental effects and endocrine system disruption. Today, there is increasing concern about the long-term effects of anthropogenic chemicals such as polychlorinated biphenyls (PCBs), pesticides and plasticizers, among others, found in water, food, air and the materials around us. An increasing number of wildlife species are having reproductive difficulties, and there is current concern about declining sperm counts in human males.
Safe environmental concentrations of toxicants are those that allow humans and indigenous organisms in nature to complete their life cycles unimpaired. Toxicity tests that include exposure through the entire life cycle are the most useful tools for such measurements but can be too long and costly to be applied in most toxicity assessments. As a result, several short-term tests have been developed to estimate chronic toxicity, representing compromises of speed, sensitivity and cost. Most measurements of toxicity today rely on these short-term tests to indicate concentrations above which toxic effects are expected.
Daphnia are widely used in testing for aquatic toxins because of their rapid clonal reproduction, ecological importance, and sensitivity to their chemical environment. Toxicity tests using Daphnia have typically been used to detect changes in survivorship and fecundity.
Cladoceran zooplankton such as Daphnia can employ a reproductive strategy known as cyclical parthenogenesis in which one generation of sexual reproduction is interspersed with many generations of asexual reproduction. The ability to alternate life history strategy allows cladocerans such as Daphnia to achieve a high reproductive rate asexually when conditions are favorable, and to produce offspring sexually for survival when the environment becomes unsuitable. Under favorable environmental conditions, Daphnia reproduce asexually by producing eggs that hatch into female offspring that, in turn, asexually produce eggs that also hatch into female offspring, and so on. Populations can achieve high growth rates during the asexual phase as females mature in 8 to 11 days. The Daphnia population in Lake Mendota, Madison, Wis., is typically entirely female with occasional males and sexual females in late summer.
Sexual reproduction is initiated when females produce males and haploid resting eggs under conditions of stress such as crowding, food scarcity, low temperatures, short photoperiod, or chemical cues emitted by predators. Males mate with sexual females to produce resting eggs that can persist in a dormant state for years, allowing the population to survive hard times. Fertilized zygotes develop into embryos that enter diapause. These embryos are contained in a durable ephippium and can remain viable for years in sediments before hatching in response to environmental cues. Production of ephippia can be essential to maintain a Daphnia population in an environment that periodically becomes uninhabitable.
Bioassays that employ Daphnia are used to monitor and give a rough indication of the level of contamination in waters, and to test a specific chemical to predict the risk posed to biological communities. There are several standard assays that presently use Daphnia for measuring the toxicity of chemical substances in an aquatic sample. One such bioassay has been developed by the U.S. Environmental Protection Agency (EPA) to assess the relative toxicity of effluents and surface waters (U.S. EPA, "Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms"(3d ed.), Section 13, Daphnid, Ceriodaphnia dubia, Survival and Reproduction Test, Method 1002.0, Lewis et al. (eds.), Environmental Monitoring Systems Laboratory, Cincinnati, Ohio (EPA/600/4-91/002, July 1994)). The EPA bioassay employs neonates (&lt;24 hours old) of Ceriodaphnia dubia during a three-brood, 7-day static renewal test, with test results measured in terms of survival and reproduction.
Another bioassay that is currently used is a 21-day test provided by the American Society for Testing and Materials (ASTM) to examine the toxicity of a water sample or other material (ASTM Standards on Aquatic Toxicology and Hazard Evaluation, Standard Guide for Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna, Method E 1193-87, Philadelphia, Pa. (ASTM PCN 03-547093-16, May 1988). The ASTM Daphnia assay is labor-intensive and the organisms are grown at high food levels which involves regularly changing water to keep the organisms well fed and maximally reproducing. A single organism is placed into each of ten separate vials, the test is run for 21 days, and the endpoint measurement is the total number of surviving organisms. Under these conditions, the Daphnia reproduce female offspring by asexual reproduction.
A drawback of the EPA and ASTM Daphnia bioassays is that the assays only consider the effects on Daphnia during the asexual phase of their life cycle. In those bioassays, the animals are grown under conditions that optimize growth and asexual reproduction. However, projections of the species, community or ecosystem level risk posed by water-borne contaminants based on bioassays of asexual reproduction may ignore serious effects on reproductive strategy. Such bioassays can miss or make false predictions about the real effects of toxic chemicals in the environment because they do not measure toxic effects during a fundamental part of the life cycle of the model organism. Many invertebrates such as Daphnia with complex, multi-stage life histories may show variable sensitivity at different points in their development and over the course of an annual cycle. Toxicity can have entirely different consequences if it coincides with sub-optimal environmental factors. Effects that occur during crucial periods such as the sexual production of dormant stages, may have disproportionate consequences at the population level.
Other disadvantages of the EPA and ASTM assays are that they tend to provide inconsistent and highly variable, irreproducible results within and between laboratories, do not measure the effects of contaminants on production of males, and provide no information on sex ratio or on developmental changes.
Daphnia bioassays have been disclosed that subject the organism to stress to measure survivorship. See for example, U.S. Pat. Nos. 5,481,815 (Murphy) and 5,169,777 (Haley) which describe a 48-hour toxicity bioassay using Daphnia magna neonates. Drawbacks of such assays are that they measure only strong (e.g., acute) effects of a contaminant and fail to detect subtler effects that may be ecologically important, and give no information on male production or sex ratio or resting egg production. Anthropogenic chemicals interact with developmental pathways of many different animals, and invertebrates such as Daphnia which have alternate life history patterns may be susceptible to different chemical interactions during its two stages of reproduction. Such effects would not be detected in a 48-hour bioassay. Furthermore, a 48-hour bioassay would not detect the consequences of toxic effects on sexual reproduction of Daphnia which can appear in depressed hatching of resting eggs.
Many pesticides, toxic xenobiotics such as some PCBs and dioxins, and common industrial chemicals such as nonylphenol and phthalates, affect and disrupt the endocrine system of an animal (i.e., endocrine disrupter). Such chemical substances can function as estrogen mimics and are implicated as agents that interfere with male development in wildlife populations such as alligators, sea gulls, turtles, salmon and trout. Endocrine disrupters effect the life history of Daphnia to trigger or suppress the production of males and resting eggs, and/or cause visible developmental deformities in the offspring. For example, low levels of the plasticizer nonylphenol have been shown to reduce resting egg production, and cause a characteristic deformity by preventing normal development from the embryo to the neonate stage. However, there is no bioassay currently available to quickly and reliably test for the presence of endocrine disrupters in a water sample.
Accordingly, an object of the invention is to provide a bioassay that is relatively easy to perform, fast, highly accurate and sensitive, and provides ecologically meaningful test information to evaluate environmental toxicity. Another object is to provide a test assay that is highly consistent within and between laboratories, and is easy to perform. Another object is to provide a bioassay and test kit for detecting toxic substances in a sample and to relate such toxicity to a particular type of chemical agent. Yet another object is to provide a bioassay that can be used to detect the presence of an endocrine disrupter, including estrogen mimics, in a water sample, and to test the activity of a chemical substance for activity as an endocrine disrupter.